Eukaryotic RNA polymerase I (Pol I) transcribes ribosomal RNA, key components of ribosomes. Pol I transcription accounts for the majority of the total RNA in cells, and its upregulation in human cells is a hallmark of cancer, making it an attractive therapeutic target for cancer. There are major gaps in our understanding of (1) the structural organization and architecture of Pol I transcription complexes, (2) the mechanism of Pol I transcription initiation and the molecular function of Pol I initiation factors, and (3) the regulation of Pol I activity by tumor suppressors and oncogenes. My primary career goals are to become an independent investigator at a top academic institution in the US, to establish a successful and well-funded research program, and to become a leader in the Pol I and transcription fields. To accomplish these goals, I will follow a career development plan that will expand my scientific and leadership skills through participation in grant writing, leadership, and laboratory management workshops, and by attending scientific meetings with topics on transcription, ribosome biogenesis, proteomics, and cancer. The long-term objective of this research proposal is to understand the mechanism of Pol I transcription and how it is dysregulated in cancer. The rationale is that understanding the Pol I transcription mechanism at the most basic and fundamental levels will translate to a better understanding of the connection between Pol I and cancer, leading to new cancer therapeutic strategies. My proposed research will use a conceptually and technically innovative cross-organismal and interdisciplinary approach that employs a combination of bioinformatics, computational, molecular, biochemical, genetic, proteomic, and structural methods in the yeast and human systems. My recent breakthrough and paradigm shifting discovery of a TFIIB-related Pol I initiation factor represents a significant and substantive departure from the status quo in the Pol I field. Guided by these preliminary studies, I will test three specific aims: (1) Determine the molecular architecture of te eukaryotic RNA polymerase I preinitiation complex, (2) Determine the molecular functions of the Pol I-specific initiation factors, and (3) Determine the mechanism by which the tumor suppressor p53 downregulates Pol I transcription. To accomplish these aims, I will use three well-established and complementary approaches to identify and map novel Pol I interactions in the context of a normal preinitiation complex environment. These methods include combined chemical crosslinking and mass spectrometry, site-specific UV-photocrosslinking, and site-specific hydroxyl radical protease footprinting that are new to the Pol I field. To complement these studies and to test the functional relevance of the observed interactions, I will use structural modeling and a combination of molecular, genetic, and biochemical assays to identify Pol I factor functions conserved from yeast to human. The proposed research is significant because it will lead to a detailed description of the Pol I transcription mechanism and provides a conceptual framework for understanding the link between Pol I and cancer. Ultimately, this work will illuminate key steps in Pol I transcription that can be used in targeted therapies.